Plasmodium Falciparum Dihydrofolate Reductase-thymidylate Synthase Research Articles

Antimalarial and Antileishmanial Flavonoids from Calendula officinalis Flowers

Calendula officinalis L. (Asteraceae), commonly known as English or pot marigold, is an herbaceous plant with edible flowers. In this study, UPLC-ESI-MS/MS analysis was used for tentative identification of compounds in marigold flower methanol extract (MFE). In addition, RP-HPLC-DAD analysis was used to quantify the flavonoids hesperidin and rutin in MFE. The antileishmanial potentials of the crude extract and compounds were evaluated against Leishmania major promastigotes and amastigotes. Further, in vivo 4-day antimalarial testing of the extract and compounds was carried out at doses of 25 mg kg−1 per day using mice infected with ANKA strain of Plasmodium berghei, following standard procedure. Molecular docking studies were carried out to assess the binding mode of flavonoids against the vital targets of L. major, including pteridine reductase 1 and farnesyl diphosphate synthase enzymes. The in silico antimalarial potentials of flavonoids were evaluated against wild-type Plasmodium falciparum dihydrofolate reductase-thymidylate synthase and phosphoethanolamine methyltransferase enzymes. Twenty compounds were tentatively identified by UPLC-ESI-MS/MS analysis of MFE, of which, seven flavonoids, six saponins, three phenolic acids, three fatty acids, and a triterpene glycoside were identified. MFE phytochemical analysis revealed that hesperidin content was 36.17 mg g−1 extract, that is, 9.9-fold their content of rutin (3.65 mg g−1 extract). The method was validated to ensure reproducibility of the results. The tested samples exhibited antileishmanial potentials against L. major promastigotes, with IC50 values of 98.62, 118.86, and 104.74 ng µL−1 for hesperidin, rutin, and MFE, respectively. Likewise, hesperidin showed inhibitory potentials against L. major amastigote with an IC50 value of 108.44 ± 11.2 µM, as compared to miltefosine. The mean survival time, parasitemia, and suppression percentages showed similar results for the three samples against ANKA strain of P. berghei. The docking studies showed good binding affinities of rutin and hesperidin with numerous H-bonding and van der Waals interactions. Marigold flowers are nutraceuticals, presenting important sources of bioactive flavonoids with potential against neglected tropical diseases.

Palladium-catalyzed facile synthesis of imidazo[1,2-a]pyridine-flavone hybrids and evaluation of their antiplasmodial activity

An efficient cross-coupling reaction between imidazo[1,2-a]pyridine derivatives and 6-bromoflavones has been well established. This reaction proceeds through a Palladium-catalyzed cross-coupling reaction to provide imidazo[1,2-a]pyridine-flavone hybrids in good to excellent yield. Short reaction time, high yield, and wide substrate scope are the major advantages of this method. Using SYBR Green I assay, these hybrid molecules were examined for anti-plasmodial activity against Chloroquine-sensitive 3D7 and Chloroquine-resistant K1 strains of Plasmodium falciparum. The compounds 9o {IC50s (µM) 1.983D7, 1.98K1} and 9p {IC50s (µM) 1.923D7, 5.45K1} were found to be the most potent anti-plasmodial compounds in the series. Also, all these compounds were found to be non-cytotoxic towards Vero cells with their CC50s > 100 µM. We have conducted microscopic examination experiments on compounds 9o and 9p, which resulted in a drastic impact on parasite growth of the malaria parasite. The interaction of these two potent hybrids was also examined in the binding site of wild-type Pf-DHFR-TS (Plasmodium falciparum dihydrofolate reductase-thymidylate synthase) using molecular docking studies. The computational ADME studies of the hybrid compounds confirmed their significant physicochemical, pharmacokinetic, and drug-likeness properties confirming these hybrids as a new oral antimalarial agent. As a result, these new hybrid compounds could be valuable in the creation of future antimalarial drugs.

Antimalarial potential, LC–MS secondary metabolite profiling and computational studies of Zingiber officinale

Malaria is among the top-ranked parasitic diseases that pose a threat to the existence of the human race. This study evaluated the antimalarial effect of the rhizome of Zingiber officinale in infected mice, performed secondary metabolite profiling and detailed computational antimalarial evaluation through molecular docking, molecular dynamics (MD) simulation and density functional theory methods. The antimalarial potential of Z. officinale was performed using the in vivo chemosuppressive model; secondary metabolite profiling was carried out using liquid chromatography–mass spectrometry (LC–MS). Molecular docking was performed with Autodock Vina while the MD simulation was performed with Schrodinger desmond suite for 100 ns and DFT calculations with B3LYP (6-31G) basis set. The extract showed 64% parasitaemia suppression, with a dose-dependent increase in activity up to 200 mg/kg. The chemical profiling of the extract tentatively identified eight phytochemicals. The molecular docking studies with plasmepsin II and Plasmodium falciparum dihydrofolate reductase-thymidylate synthase (PfDHFR-TS) identified gingerenone A as the hit molecule, and MMGBSA values corroborate the binding energies obtained. The electronic parameters of gingerenone A revealed its significant antimalarial potential. The antimalarial activity elicited by the extract of Z. officinale and the bioactive chemical constituent supports its usage in ethnomedicine. Communicated by Ramaswamy H. Sarma

Quantitative Structure-Activity Relationship, Structure-based Design, and ADMET studies of pyrimethamine and cycloguanil analogs inhibitors of Plasmodium falciparum dihydrofolate reductase-thymidylate synthase (PfDHFR-TS).

Quantitative Structure-Activity Relationship, Structure-based Design, and ADMET studies of pyrimethamine and cycloguanil analogs inhibitors of Plasmodium falciparum dihydrofolate reductase-thymidylate synthase (PfDHFR-TS).

Substituted 3-styryl-2-pyrazoline Derivatives as an Antimalaria: Synthesis, in vitro Assay, Molecular Docking, Druglikeness Analysis and Admet Prediction

The synthesis, in vitro antimalarial assay, molecular docking, drug-likeness analysis, and ADMET prediction of substituted 3-styryl-2-pyrazoline derivatives as antimalaria have been conducted. The synthesis of N-phenyl (1a‒3a) and N-acetyl-substituted (1b‒3b) 3-styryl-2-pyrazolines was carried out using dibenzalacetone derivatives and hydrazine hydrate or phenylhydrazine. An in vitro antimalarial assay was conducted against the chloroquine-sensitive Plasmodium falciparum 3D7 strain, while molecular docking was performed toward the crystal protein of Plasmodium falciparum dihydrofolate reductase-thymidylate synthase (PfDHFR-TS) (PDB ID: 1J3I). Furthermore, the prediction of drug-like properties was determined by assessing Lipinski’s rules, and the pharmacokinetic parameters were also studied in-silico, including absorption, distribution, metabolism, excretion, and toxicity (ADMET). The in vitro assay showed that 3a (IC50 0.101 µM) has excellent antimalarial activity, followed by 2a (0.177 µM), and 1b (0.258 µM). Molecular docking has supported the in vitro assay by showing the lowest CDOCKER energy for 3a (‒56.316 kcal/mol), then 2a (‒51.2603 kcal/mol), and 1b (‒48.8774 kcal/mol). The drug-like properties showed that all of the prepared compounds were acceptable based on Lipinski’s rules and predicted to be potentially orally bioavailable. The ADMET analysis provided information that 3a and 2a could be proposed as the best lead antimalarial drugs with further modification to reduce the lipophilicity and toxicity properties.

Key interactions of pyrimethamine derivatives specific to wild-type and mutant P. falciparum dihydrofolate reductase based on 3D-QSAR, MD simulations and quantum chemical calculations

Plasmodium falciparum dihydrofolate reductase-thymidylate synthase (PfDHFR-TS) is an important target enzyme in malarial chemotherapy. An understanding of how novel inhibitors interact with wild-type (wtPfDHFR), quadruple-mutant (qmPfDHFR), and human (hDHFR) enzymes is required for the development of these compounds as antimalarials. This study is focused on a series of des-Cl and m-Cl phenyl analogs of pyrimethamine with various flexible 6-substituents. The interactions of these compounds with DHFR enzymes were investigated by 3 D-QSAR, MD simulations, MM-PBSA, and DFT calculations. CoMFA and CoMSIA models were developed with good predictive abilities for wtPfDHFR and qmPfDHFR. For hDHFR, CoMSIA models combined with clogP descriptor were successfully derived. Binding free energy using MM-PBSA and comparison of per residue decomposition energy analyses with the DFT method at M06-2X/6-31G ++(d,p) level of theory indicated that Asp54 and Phe58 play important roles in the binding of the most potent compound in the series (compound 27) with both wtPfDHFR and qmPfDHFR, whereas Arg59 and Arg122 were additionally found to interact with this inhibitor in qmPfDHFR. For hDHFR, the residues Glu30 and Phe34 but not Arg70, equivalent to Asp54, Phe58, and Arg122 in PfDHFR, also play role in compound 27 binding through strong hydrophobic interactions (Phe34) and hydrogen bond network with Glu30, Ile7, and Val115. From the key interactions identified in the DHFR-inhibitor complexes, a general scheme is proposed for designing new inhibitors selective for PfDHFR that is important for the development of novel antifolate antimalarials. Communicated by Ramaswamy H. Sarma

In Silico Studies of Bioactive Compounds Selected from Four African Plants with Inhibitory Activity Against Plasmodium falciparum Dihydrofolate Reductase-Thymidylate Synthase (pfDHFR-TS)

In Silico Studies of Bioactive Compounds Selected from Four African Plants with Inhibitory Activity Against Plasmodium falciparum Dihydrofolate Reductase-Thymidylate Synthase (pfDHFR-TS)

Synthesis and Docking Study of 2–Aryl-4,5-diphenyl-1<i>H</i>-imidazole Derivatives as Lead Compounds for Antimalarial Agent

Series of 2-aryl-4,5-diphenyl-1H-imidazole derivatives of 2-(4-hydroxy-3-methoxyphenyl)-4,5-diphenyl-1H-imidazole (1), 2-(4,5-dimethoxyphenyl)-4,5-diphenyl-1H-imidazole (2) and 2-(4-methoxyphenyl)-4,5-diphenyl-1H-imidazole (3) were produced and evaluated for their in vitro antimalarial activities against the chloroquine-sensitive Plasmodium falciparum 3D7 strain. A molecular docking study was also carried out against the crystal protein of Plasmodium falciparum dihydrofolate reductase-thymidylate synthase (PfDHFR-TS) (PDB ID: 1J3I.pdb) to predict the interaction between the compounds and protein. The physicochemical and pharmacokinetic parameters were computationally performed to predict the parameters of the absorption, distribution, metabolism, excretion, and toxicity (ADMET). Imidazoles were synthesized from aryl aldehyde derivatives with benzyl and ammonium acetate in glacial acetic acid using microwave-assisted-organic synthesis. Compounds 1, 2, and 3 were produced in 64.33, 50.56, and 70.55% yields, respectively. The IC50 of compounds 1, 2, and 3 against chloroquine-sensitive Plasmodium falciparum 3D7 strain was found to be 1.14, 5.28, and 2.42 µM, respectively. The molecular docking study agreed with the in vitro data by showing the lowest CDOCKER energy for compound 1 (-47.48 kcal/mol), followed by 3 (-43.79 kcal/mol) and 2 (-41.47 kcal/mol). The physicochemical and pharmacokinetic parameters showed that imidazoles 1, 2, and 3 obeyed Lipinski rules of five to propose as lead compounds for the antimalarial agents.

Molecular Docking Studies of (2E)-1(1-Methyl-1H-Indol-3-yl)-3-Phenyl-Prop-2-en-one) as Antimalarial and Its Synthesis using Dimethylsulfate

Malaria is a disease with high prevalence in tropical and subtropical areas. The case of parasite resistance to proguanil, an antimalarial drug, has prompted the discovery of new drug candidates through molecular docking studies. In this study, we used the derivative of chalcone (2E)-1-(1-methyl-1H-indol-3-yl)-3-phenyl-prop-2-en-1-one as a test ligand and some comparison ligands to see its interaction with Plasmodium falciparum dihydrofolate reductase - thymidylate synthase (PfDHFR-TS) enzyme. Furthermore, the compound (2E)-1-(1-methyl-1H-indol-3-yl)-3-phenyl prop-2-en-1-one was synthesized by reacting (2E)-1-(1H-indol-3-yl)-3-phenyl prop-2-en-1-one and dimethyl sulfate through an N-alkylation reaction. The (2E)-1-(1-methyl-1H-indol-3-yl)-3-phenyl-prop-2-en-1-one has the higher ΔG bind (-7.53 kcal/mole) than native ligan WR99210(-8.83 kcal/mole), but has the lower than proguanil (-6.75 kcal/mole) and (E)-3-(3,4-dimethoxy phenyl)-1-(2-hydroxy-4-methoxy-5-(prenyl)phenyl)-prop-2-en-1-one (-7.29 kcal/mole). However, there was only pi interaction of amino acid residue of the PfDHFR-TS ligand complexes (PHE58, ILE112, LEU119, ALA16, ILE14), in contrast to the WR99210 and proguanil complexes. This is similar to the interaction of the (E)-3-(3,4-dimethoxyphenyl)-1-(2-hydroxy-4-methoxy-5-(prenyl)phenyl)-prop-2-en-1-one-PfDHFR-TS complex which is active as an antimalarial in vitro, so that the test ligand still has a chance to be used as an antimalarial candidate. The compound (2E)-1-(1-methyl-1H-indol-3-yl)-3-phenyl prop-2-en-1-one was successfully synthesized with 41.55% yield.

Design, Synthesis and Biological Evaluation of Aminoalkylated Chalcones as Antimalarial Agent

Aminoalkylated chalcone compounds (4a-4c) have been designed using Quantitative Structure-Activity Relationship (QSAR) analysis, synthesized and evaluated for their in vitro antimalarial activity. The best QSAR model obtained was log IC50 = 705.132 (qC7) - 65.573 (qC3) - 24.845 (qC4) - 4.634 (qC13) - 220.479 and statistical analysis showed R2 of 0.937, suggesting that the QSAR model was able to predict the actual antimalarial activity by 93.7% accuracy. The addition of secondary amines to the chalcone compounds was successfully carried out using the Mannich reaction, which was confirmed by spectroscopic analysis. The in vitro antimalarial activity of the synthesized compounds were screened against the 3D7 strain of Plasmodium falciparum (CQ sensitive). All of the compounds exhibited strong activity with IC50 values ranging from 0.54 ± 0.649 to 1.12 ± 0.369 µM. The molecular docking studies investigated interactions of the prepared compounds to the binding site of wild-type Plasmodium falciparum dihydrofolate reductasethymidylate synthase (Pf-DHFR-TS) (PDB ID: IJ3I) and quadruple mutant Pf-DHFR-TS (PDB ID: IJ3K). Some hydrogen bond and π – π interactions were observed with the side chain of Ala16, Asp54, Cys15, Leu164, Tyr170, and Met55 in both the wild and mutant Pf-DHFR types. It has also been found that all the tested compounds were obeyed the Lipinski’s rule. This study proposed that compound 4b can be developed as the new lead of the antimalarial agent.

Antimalarial effect of cell penetrating peptides derived from the junctional region of Plasmodium falciparum dihydrofolate reductase-thymidylate synthase

Dihydrofolate reductase-thymidylate synthase of Plasmodium falciparum (PfDHFR-TS) is an important target of antifolate antimalarial drugs. However, drug resistant parasites are widespread in malaria endemic regions. The unique bifunctional property of PfDHFR-TS could be exploited for the design of allosteric inhibitors that interfere with the active dimer conformation. In this study, peptides were derived from the junctional region (JR) of PfDHFR-TS amino acid sequence in the αj1 helix (JR-helix) and the DHFR domain that is necessary for interaction with αj1 helix (JR21). Five peptides were synthesized and tested for inhibition of PfDHFR-TS enzyme by Bacterial inhibition assay (BIA) based on the growth of an E. coli DHFR and TS knockout complemented with a recombinant plasmid expressing PfDHFR-TS enzyme. Significant inhibition was observed for JR21 and JR21 conjugated to cell-penetrating octa-arginine peptide (rR8-JR21) with 50 % inhibitory concentration (IC50) of 3.87 and 1.53 μM, respectively. The JR-helix and rR8-JR-helix peptides were inactive. JR21 and rR8-JR21 peptides showed similar growth inhibitory effects on P. falciparum NF54 parasites cultured in vitro. Treatment with rR8-JR21 delayed parasite development, in which an accumulation of ring stage parasites was observed after 12 h of culture. Minimal red blood cell (RBC) hemolysis was observed at the highest dose of peptide tested. The most potent peptide rR8-JR21 not only compromised the development of the P. falciparum, but also inhibited the parasite growth and has low hemolytic effect on human RBCs.

Spectroscopic characterization of hydroxyquinoline derivatives with bromine and iodine atoms and theoretical investigation by DFT calculations, MD simulations and molecular docking studies

In the present work, spectroscopic characterization of 5,7-dibromo-8-hydroxy quinoline (DBHQ(1)) and 5,7-diiodo-8-hydroxy quinoline (DIHQ(2)) has been obtained theoretically and experimentally. On the basis of potential energy distribution of vibrational modes, complete assignments of wavenumbers were performed yielding good agreement between experimental and theoretical wavenumbers. The HOMO-LUMO plots in the title molecules show the charge transfer in the molecular system through the conjugated paths. The electrophilic and nucleophilic sites are revealed from the molecular electrostatic potential maps. Experimental investigation encompassed spectroscopic characterization by FT-IR and Raman techniques while computational studies included DFT calculation, molecular dynamics (MD) simulations and molecular docking studies. MD simulations provided insights into the reactivity with water and with selected proteins. The molecular docking studies reveal that the ligands bind at the active site of the macromolecule and could restrict or block the functioning of Plasmodium falciparum dihydrofolatereductase-thymidylate synthase (PfDHFR-TS), there by acting as antiprotozoal agents. Drug activity and binding affinity of halogen positional changes in the title molecules with target protein were reported and this study thesaurus the effects of bio-efficiency in the molecule.

Microwave assisted synthesis, antimalarial screening and structure–activity-relationship exploration of some phenylthiazolyl-triazine derivatives against dihydrofolate reductase

In this study, a microwave-assisted methodology was first attempted to facilitate the synthesis of hybrid phenylthiazolyl-triazine derivatives. These two nuclei were clubbed together based on the structural requirement of existing antimalarial antifolates. A comparative analysis revealed that compounds synthesized using microwave-assisted procedure gave better yield and minimized the reaction time with respect to the conventional procedure. Hybrid compounds were screened for their in vitro antimalarial activity against chloroquine-sensitive (3D-7) strain of Plasmodium falciparum at 5 µg/mL and 50 µg/mL dose level. An insight into the structure–activity-relationship of the synthesized compounds was gained by docking them in the crystal structure of wild type Plasmodium falciparum dihydrofolate reductase-thymidylate synthase.

Electrostatic Channeling in P. falciparum DHFR-TS: Brownian Dynamics and Smoluchowski Modeling

We perform Brownian dynamics simulations and Smoluchowski continuum modeling of the bifunctional Plasmodium falciparum dihydrofolate reductase-thymidylate synthase (P. falciparum DHFR-TS) with the objective of understanding the electrostatic channeling of dihydrofolate generated at the TS active site to the DHFR active site. The results of Brownian dynamics simulations and Smoluchowski continuum modeling suggest that compared to Leishmania major DHFR-TS, P. falciparum DHFR-TS has a lower but significant electrostatic-mediated channeling efficiency (∼15–25%) at physiological pH (7.0) and ionic strength (150 mM). We also find that removing the electric charges from key basic residues located between the DHFR and TS active sites significantly reduces the channeling efficiency of P. falciparum DHFR-TS. Although several protozoan DHFR-TS enzymes are known to have similar tertiary and quaternary structure, subtle differences in structure, active-site geometry, and charge distribution appear to influence both electrostatic-mediated and proximity-based substrate channeling.

In silico screening of antifolate based novel inhibitors from Brucea mollis Wall. ex kurz against quadruple mutant drug resistant PfDHFR.

Plasmodium falciparum is the most lethal form of the genus Plasmodium which causes malaria, a 'disease of antiquity'. Globally it affects the health and socio-economic development of a large population especially in Sub-Saharan Africa and Southeast Asia. The Plasmodium falciparum dihydrofolate reductase-thymidylate synthase (PfDHFR-TS) is an important target of antimalarial drugs. Mutations at the active site of PfDHFR have resulted in decrease drug binding affinity of DHFR-inhibitors. In the present study we selected ten compounds of Brucea mollis Wall. Ex kurz and checked for their drug likeness using various computational tools and potential interactions with PfDHFR by molecular docking study. Soulameanone, a quassinoid of Brucea mollis Wall. Ex kurz showed better binding affinity when compared to pyrimethamine for both wild and quadruple mutant drug resistant PfDHFR. In addition, similar isomers of soulameanone were screened for their drug likeness and to study their interactions with PfDHFR. Twenty three compounds showed better binding affinity compared to soulameanone.

In silico Analysis of 2, 4-Substituted Heterocycles and Glutamic Acid Containing Antifolates as Inhibitors of Malarial (Plasmodium falciparum) Protein, Dihydrofolate Reductase-Thymidylate Synthase

Plasmodium falciparum dihydrofolate reductase-thymidylate synthase (PfDHFR-TS) function is effectively inhibited by antifolates. The binding affinity of antifolates to PfDHFR-TS is reduced due to mutations in its active site. In the present study, 33 analogues of Methotrexate (MTX), Trimetrexate (TMX), Raltitrexed (RTX) and Pemetrexed (PTX) were designed and evaluated for interaction with PfDHFR-TS by in silico methods. Analyses of drug candidates were performed by generating their docking complexes with quadruple mutant crystal structure of PfDHFR-TS using Molecular Operating Environment (MOE). Initially eight top scoring complexes and then finally two (MTX04 and PTX03) were found suitable for further optimization based on interaction pattern with active site amino acids. Analyses of structural characteristics, binding energy calculations and interaction patterns of MTX04 and PTX03 with DHFR and TS domains respectively as best drug candidates. The comparative docking studies of these two compounds with human proteins provided a strong evidence of selectivity for MTX04 as effective antimalarial drug candidate. It is considered that the drug will inhibit the activity of folate pathway and it will be effective source to control malaria.

Insights into the role of the junctional region of Plasmodium falciparum dihydrofolate reductase-thymidylate synthase

BackgroundPlasmodium falciparum dihydrofolate reductase-thymidylate synthase (pfDHFR-TS) is a well-defined target of anti-malarial drug, such as pyrimethamine and cycloguanil. Emergence of malaria parasites resistant to these drugs has been shown to be associated with point mutations of the gene coding for the target enzymes. Although the 3D-structure of P. falciparum bifunctional pfDHFR-TS has been reported previously, relatively little is known about the interactions between the pfDHFR and pfTS domains and the roles of the junctional region that links the two domains together. Therefore, a thorough understanding of the interaction of the two domains and the role of the junctional region of this target is important as the knowledge could assist the development of new effective anti-malarial drugs aimed at overcoming drug-resistant malaria.MethodsA system was developed to investigate the interaction between pfDHFR and pfTS domains and the role of the junctional region on the activity of the recombinant pfTS. Based on the ability of co-transformed plasmids coding for pfDHFR and pfTS with truncated junctional region to complement the growth of TS-deficient Escherichia coli strain χ2913recA(DE3) on minimum media without thymidine supplementation, active pfTS mutants with minimal length of junctional region were identified. Interactions between active pfDHFR and the pfTS domains were demonstrated by using a bacterial two-hybrid system.ResultsUsing TS-deficient E. coli strain χ2913recA(DE3), the authors have shown for the first time that in P. falciparum a junctional region of at least 44 amino acids or longer was necessary for the pfTS domain to be active for the synthesis of thymidylate for the cells. Truncation of the junctional region of the bifunctional pfDHFR-TS further confirmed the above results, and suggested that a critical length of the junctional peptide of pfDHFR-TS would be essential for the activity of TS to catalyze the synthesis of thymidylate.ConclusionThe present study demonstrated the interactions between the pfDHFR and pfTS domains of the bifunctional pfDHFR-TS, and revealed that the junctional region linking the two protein domains is essential for the expression of catalytically active pfTS domain. The findings could be useful since inhibition of the pfDHFR-TS domain-domain interaction could form a basis for the development of new anti-malarial drugs based on targeting the non-active site region of this important enzyme.

Exploring QSAR, Pharmacophore Mapping and Docking Studies and Virtual Library Generation for Cycloguanil Derivatives as PfDHFR-TS Inhibitors

Resistance of available antimalarial drugs against Plasmodium species is one of the major problems of malaria control in the developing world. In the present study, we have performed QSAR, pharmacophore mapping and molecular docking studies of cycloguanil derivatives as Plasmodium falciparum dihydrofolate reductase thymidylate synthase (PfDHFR-TS) inhibitors to explore essential features required for the antimalarial activity and important interaction patterns between the enzyme and ligands for the design of new potent PfDHFR-TS inhibitors. The QSAR studies have been carried out using topological parameters along with thermodynamic and structural descriptors. Acceptable values of internal and external validation parameters for the developed QSAR models confirm acceptability of the models. Pharmacophore mapping revealed that two hydrogen bond donor (HBD) features and a hydrophobic feature (HYD) are important parameters for PfDHFR-TS inhibitory activity. The docking studies suggest that the PfDHFR-TS inhibitors interact with Asp54, Ile14, Ile164, ser108, Ser111, Tyr170, Met55, Ala16, Thr185, Leu46, Cys15, Phe58, Ile112, Trp48, Tyr57 and Leu119 amino acid residues. The QSAR, pharmacophore and docking studies inferred that i) branching of the substituents at R1 and R2 positions should be less (small alkyl chain substituents are favored); ii) the electronegativity of the molecules should be high but within some limit; iii) the size and volume of the molecules should be high; iv) molecules should be flexible enough; v) R configuration at C6 position of the triazine ring favors the inhibitory binding affinity; vi) the substituents of the phenyl ring at 3, 4 and 5 position of the phenyl ring should be small hydrophobic groups. Based on these studies, we have designed a library of cycloguanil derivatives with good in silico predicted PfDHFR-TS inhibitory activity.

Subunit complementation of thymidylate synthase in Plasmodium falciparum bifunctional dihydrofolate reductase-thymidylate synthase

Thymidylate synthase of Plasmodium falciparum dihydrofolate reductase-thymidylate synthase ( PfDHFR-TS) functions as a dimeric enzyme with extensive contact between the two TS domains. Structural data of PfDHFR-TS shows that the formation of the two TS active sites involves contribution of the amino acid residues from both TS domains. Arg-470 donated from the adjoining domain is shown to hydrogen-bond to dUMP, while Cys-490 is a key nucleophile for TS catalysis by attacking C-6 of dUMP. However, mutants of the two series could complement one another, giving rise to active enzyme. By means of subunit complementation assay using Arg-470 and Cys-490 mutants, it is shown that co-transformants of both TS-inactive Arg-470 and Cys-490 mutants can complement the growth of thymidine auxotroph χ2913RecA(DE3) by formation of a functional TS heterodimer contributing from both Arg-470 and Cys-490 mutant subunits. 6-[ 3H]-FdUMP thymidylate synthase activity assay further elaborate the essence of restoration of TS activity. The TS k cat value of the R470D + C490A heterodimer is decreased by half from that of the wild-type PfDHFR-TS. However, the K m values for dUMP and CH 2H 4folate of the R470D + C490A heterodimer are similar to those of wild-type enzyme, indicating that the catalytic efficiency of the functional TS from the R470D + C490A heterodimer is similar to the wild-type TS enzyme in P. falciparum DHFR-TS.

Insights into antifolate resistance from malarial DHFR-TS structures.

Plasmodium falciparum dihydrofolate reductase-thymidylate synthase (PfDHFR-TS) is an important target of antimalarial drugs. The efficacy of this class of DHFR-inhibitor drugs is now compromised because of mutations that prevent drug binding yet retain enzyme activity. The crystal structures of PfDHFR-TS from the wild type (TM4/8.2) and the quadruple drug-resistant mutant (V1/S) strains, in complex with a potent inhibitor WR99210, as well as the resistant double mutant (K1 CB1) with the antimalarial pyrimethamine, reveal features for overcoming resistance. In contrast to pyrimethamine, the flexible side chain of WR99210 can adopt a conformation that fits well in the active site, thereby contributing to binding. The single-chain bifunctional PfDHFR-TS has a helical insert between the DHFR and TS domains that is involved in dimerization and domain organization. Moreover, positively charged grooves on the surface of the dimer suggest a function in channeling of substrate from TS to DHFR active sites. These features provide possible approaches for the design of new drugs to overcome antifolate resistance.